The complex processes of DNA replication, recombination, and repair all require that the DNA double-helix be at least transiently unwound. In DNA replication, helicases bind to parental DNA and unwind it so that DNA polymerase may read the genetic code to synthesise a new copy or daughter strand. As the duplex DNA is unwound, a class of proteins called SSBs (derived from single-stranded DNA binding proteins) are responsible for binding single-stranded DNA (ssDNA) until it is utilised by DNA polymerase or other proteins involved in DNA recombination and repair.
DNA polymerases generally are divided according to the correspondence in their amino acid sequences into three main families with subclasses. In prokaryotes, the main distinction is made between three polymerases: polymerase I, II, and III. These polymerases differ with regard to their function in the cell and with regard to their properties. DNA polymerase I is considered to be a repair enzyme and frequently has 5′-3′ as well as 3′-5′ exonuclease activity. Polymerase II appears to facilitate DNA synthesis which starts from a damaged template strand and thus preserves mutations. Polymerase III is the replication enzyme of the cell, it synthesizes nucleotides at a high rate (ca. 30,000 per minute) and is considered to be very processive. Polymerase III has no 5′-3′ exonuclease activity.
Particular properties of polymerases are desirable depending on the application. For example, in PCR, thermophilic DNA polymerases are used to perform cyclical primer-extensions at high temperature to amplify the number of copies of a DNA product. The length, quality, and quantity of this product depend on the accuracy, stability, and processivity of the DNA polymerase.
In vivo, replicative DNA polymerases are made more processive by their interactions with accessory proteins, like SSBs, at the replication fork. SSBs are essential proteins that bind tightly and cooperatively to ssDNA during replication to remove adventitious secondary structures and protect the exposed DNA from endogenous nucleases. Historically, SSBs have sometimes been loosely referred to as “helix-destabilising proteins” because they can reduce the stability or “melt” some duplex DNAs. It should be emphasised that SSBs do not unwind dsDNA, rather, they bind and stabilise the ssDNA conformation as it becomes available either enzymatically via helicases or by binding ssDNA “bubbles” or the transiently frayed 5′ or 3′ ends of an otherwise duplex DNA. Because SSBs must bind all available ssDNA as it becomes accessible, they are highly abundant.
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While the present disclosure is susceptible to various modifications and alternative forms, specific example embodiments have been shown in the figures and are herein described in more detail. It should be understood, however, that the description of specific example embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, this disclosure is to cover all modifications and equivalents as defined by the appended claims.